1. Field of the Invention
The invention relates to a process for producing a polypeptide heterologous to E. coli. More particularly, the invention is directed to using organophosphate to improve yield of such polypeptides.
2. Description of Related Art
Expression of heterologous proteins by Escherichia coli, aided by the well-understood molecular biology and relative ease in genetic manipulation of the microorganism, has been very productive in both laboratory and industry. Typically, an inducible promoter (for example, the alkaline phosphatase promoter, the tac promoter, the arabinose promoter, etc.) is employed for the regulation of heterologous protein expression. The requirement of an induction event provides the researcher the opportunity to manage the timing of expression of the target protein. This ability is especially important for those heterologous proteins that are not well tolerated at high concentrations by the host. By achieving desirable cell density prior to the induction of expression, the volumetric yield of the desired protein may be maximized.
Cells cease to grow when the microorganism is deprived of a required nutrient. The limiting component may be carbon, nitrogen, phosphate, oxygen or any of the elements required by the cell. Under such conditions, the cells exit from the growth phase. A way to alleviate the culture of the stress responses caused by the nutrient limitation is to provide a feed of the lacking component. Common feeds introduced into fed-batch fermentation processes include glucose, amino acids, oxygen, etc.
In the case of cellular phosphorus (P), the requirement for phosphate supply is not surprising given that P is the fifth most abundant element in a cell behind carbon, oxygen, nitrogen, and hydrogen. Slanier, Adelberg and Ingraham, The Microbial World, 4th ed. (Prentice Hall, NJ 1976), p. 1357. Phosphorus is an essential component in numerous macromolecules such as nucleic acids, liposaccharides and membrane lipids. Furthermore, its role in the high-energy phosphoanhydride bonds makes it especially important in energy metabolism. E. coli is capable of utilizing inorganic phosphate (Pi), organophosphate or phosphonate as the primary P source. The uptake of Pi from the environment can be achieved through two transporter systems, the Pit and the Pst systems. For the organophosphates, most are non-transportable and they first need to be hydrolyzed enzymatically in the periplasm before the released Pi can be taken up by the Pi transport system(s). Only a few organophosphates are transportable, and glycerol-3-phosphate (G3P) is one such example. G3P and glycerophosphate-1-phosphate (G1P) are known as alpha-glycerophosphates. In response to Pi-limitation and carbon-limitation, E. coli is capable of taking up available intact G3P from the external environment into the intracellular compartment, where G3P is metabolized to yield needed phosphate or carbon. Wanner, “Phosphorus Assimulation and Control of the Phosphate Regulon”, in Escherichia coli and Salmonella Cellular and Molecular Biology, Neidhardt, ed., (second edition), American Society for Microbiology Press (1996), pp. 1357-1365.
Further references on G3P are Silhavy et al., J. Bacteriol., 126: 951-958 (1976) on the periplasmic protein related to the sn-glycerol-3-phosphate transport system of E. coli; Argast et al, J. Bacteriol., 136: 1070-1083 (1978) on a second transport system for sn-glycerol-3-phosphate in E. coli; Elvin et al., J. Bacteriol., 161: 1054-1058 (1985) on Pi exchange mediated by the glpT-dependent G3P transport system; Rao et al., J. Bacteriol., 175: 74-79 (1993) on the effect of glpT and glpD mutations on expression of the phoA gene in E. coli; and Elashvili et al., Appl. Environ. Microbiol., 64: 2601-2608 (1998) on phnE and glpT genes enhancing utilization of organophosphates in E. coli K-12. Further, Vergeles et al., Eur. J. Biochem., 233: 442-447 (1995) disclose the high efficiency of glycerol-2-phosphate (G2P), otherwise known as beta-glycerophosphate, and G3P as nucleotidyl acceptors in snake venom phosphodiesterase esterifications.
The current understanding of the two transport systems for the uptake of exogenous G3P in E. coli, the Ugp and GlpT transport systems, has been well summarized in the book Escherichia coli and Salmonella, Cellular and Molecular Biology edited by Neidhardt et. al. (second edition), supra, pp. 1364 referring to references 13 and 81. The Ugp operon belongs to the pho regulon. It is induced by phosphate limitation and positively regulated by phoB protein. The Ugp system is a periplasmic binding protein-dependent multi-component transport system, with ugpB encoding the periplasmic binding protein, ugpA and ugpC encoding integral membrane channel proteins, and ugpC encoding ATPase. GlpT is part of the glp system that mediates the uptake and metabolism of glycerol, G3P, and glycerol phosphoryl phosphodiesters (Lin et al., Annu. Rev. Microbiol., 30: 535-578 (1976); Chapter 20; pg 307-342 Dissimilatory Pathways for sugars, polyols and carboxylates. Escherichia coli and Salmonella, Cellular and Molecular Biology, second edition). This transport system is an anion exchanger that is known to mediate the efflux of Pi from the cytoplasm by exchange with external G3P. In a wild-type strain growing on G3P, while little Pi is released by cells taking up G3P via the Ugp system, Pi can be released into the periplasm when G3P is taken up via the GlpT system. If a repressive amount of Pi is released as a result of glpT-permease-mediated efflux, the pho regulon activity, the Ugp system included, will be shut off. Under certain conditions, GlpT is the only route for the exit of Pi from the cell by exchange with external G3P. Elvin et al., J. Bacteriol., 161: 1054-1058 (1985); Rosenberg, “Phosphate transport in prokaryotes,” p. 205-248. In B. P. Rosen and S. Silver (ed.), Ion Transport in Prokaryotes (Academic Press, Inc., New York, 1987).
When the capacities of the Ugp and the GlpT systems are compared to transport G3P, the maximal velocities of the two systems are similar. The apparent affinity for G3P is higher with the Ugp system than with the GlpT system. Likely, both systems will be able to supply enough G3P for cell growth if available in the growth medium. However, G3P transported exclusively via the Ugp system can serve as the sole source only of phosphate but not of carbon, while GlpT-transported G3P can serve as the sole source for both (Schweizer et al., J. Bacteriol., 150: 1154-1163 (1982)). The two ugp genes coding for the pho-regulon-dependent G3P transport system have been mapped (Schweizer et al., J. Bacteriol., 150: 1164-1171 (1982)), the ugp region containing these genes has been characterized (Schweizer et al., Mol. and Gen. Genetics, 197: 161-168 (1984)), and the regulation of ugp operon studied (Schweizer et al., J. Bacteriol., 163: 392-394 (1985); Kasahara et al., J. Bacteriol., 173: 549-558 (1991); Su et al., Molecular & General Genetics, 230: 28-32 (1991); Brzoska et al., “ugp-dependent transport system for sn-glycerol 3-phosphate of Escherichia coli,” p. 170-177 in A. Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright, and E. Yagil (ed.), Phosphate Metabolism and Cellular Regulation in Microorganisms (American Society for Microbiology, Washington, D.C., 1987); Brzoska et al., J. Bacteriol., 176: 15-20 (1994); and Xavier et al., J. Bacteriol., 177: 699-704 (1995)).
In wild-type strains, there exists a stable intracellular pool of G3P and it is maintained at approximately 200 μM. Internally, G3P can be synthesized by the enzymatic conversion of glycerol by glycerol kinase (encoded by glpK) to G3P when grown on glycerol as the sole carbon source, or from the reduction of the glycolytic intermediate, dihydroxyactone phosphate, by G3P synthase, the gene product of the gpsA gene, during growth on carbon sources other than glycerol. Since G3P is an important intermediate that forms the scaffold of all phospholipid molecules, internal glycerol phosphates may also be generated from the breakdown of phospholipids and triacylglycerol. As a metabolite, internal G3P may be channeled into the phospholipid biosynthetic pathway or be oxidized by G3P dehydrogenase to form dihydroxyacetone phosphate and fed into the glycolytic pathway.
In situations where the AP promoter is employed for regulating heterologous protein expression in E. coli, since induction occurs only after the medium is depleted of Pi, cells induced for AP promoter activity are typically starved for phosphate and in a declining state of health. They may have to scavenge for phosphate needed for cellular functions. Possible consequences of such phosphate scavenging may include turnover of ribosomes, lower cell energetics, and increased protease expression and proteolysis (St. John and Goldberg, J. Bacteriol., 143: 1223-1233 (1980)), potentially leading to less healthy cells with reduced capacity for protein accumulation.
Improving the metabolic state of E. coli may conceivably increase the capacity of the cell to synthesize proteins. If phosphate is fed slowly, the cells may only sense low Pi concentration in the periplasm, thereby inducing the pho regulon without being starved intracellularly for the P atom (see U.S. Pat. No. 5,304,472). There is a need for providing further methods of producing heterologous polypeptides in E. coli. 